Abstract

Mycoplasma hyopneumoniae colonise swine ciliated respiratory epithelia, leading to the development of porcine enzootic pneumonia. Heparin and other glycosaminoglycans are known to block adherence of this pathogen to porcine tracheal cilia, however adhesins with heparin-binding capacity have not been identified. Previous studies have implicated a 97 kDa surface protein of M. hyopneumoniae, P97 that appeared to be involved in the interaction of M. hyopneumoniae with swine cilia. This study aimed at identifying other potential adhesins of M. hyopneumoniae. In this study, immuno-electron microscopy and trypsin degradation analyses demonstrated that a 159 kDa protein (P159) resides on the surface of M. hyopneumoniae. Furthermore, proteomic analyses indicate that this molecule is post-translationally cleaved. In vitro, these proteins are found to be cleaved and highly expressed at all stages during the growth cycle of M. hyopneumoniae. This molecule was also shown to be immunogenic due to its reactivity with sera from pigs naturally infected with M. hyopneumoniae. Recombinant expression of P159 domains was undertaken (F1, F2, F3, and F4). Two of these domains (F3 and F4) were found to bind heparin in a dosedependent, saturable and specific manner. The Kd for this interaction was 142.37 + 22.01 nM for F3 and 75.37 + 7.34 nM for F4. Some pathogenic bacteria have been shown to bind heparin which can then bind to further components on the surface of cells, thereby acting as a bridging molecule in adhesion to host cells. Non-labeled heparin was shown to competitively inhibit this interaction with an IC50 value of 52.92 + 1.03 μg/ml for F3 and 66.63 + 1.02 μg/ml for F4. Fucoidan was also shown to competitively inhibit the binding of heparin to F3 (IC50 96.28 + 1.19 μg/ml) and F4 (IC50 36.23 + 1.14 μg/ml). Fluorescent and electron microscopic studies employing latex beads coated with P159 domains revealed that F2, F3 and F4 promoted adherence to the porcine epithelial-like cell line PK15. Additionally, F2 and F4 also mediated the uptake of the latex beads into PK15 cells. Collectively this data suggests that P159 is a good candidate for an adhesin of M. hyopneumoniae and thus may play a role in the colonization of the respiratory tract of swine. Ribosomal protein L7/L12 has been shown to play a role in the pathogenesis of a number of bacterial pathogens in vitro, and has been found to be expressed on the cell surface of some pathogenic bacteria. While work in this study revealed that the L7/L12 molecule is cytoplasmically expressed in M.hyopneumoniae, an immunoreaction with convalescent pig serum indicated the protein has immunogenic properties. The immunogenicity of the M. hyopneumoniae L7/L12 protein may make it a potential target for vaccine development.